Transcription Coactivator Mediator Subunit Med1 Is Required For The Development Of Fatty Liver In The Mouse

Nonalcoholic fatty liver disease (NAFLD), one of metabolic syndrome, is a burgeoning common chronic liver disorder with a morphological spectrum of liver pathology commencing with hepatic steatosis and steatohepatitis which may progress toward the development of cirrhosis and liver cancer, and NAFLD has become a major health concern worldwide. Identification of NAFLD in recent years raises new challenges about its effective precaution and therapy in basic and clinic research. Since the key aspects of lipid metabolism including lipogenesis, fatty acid oxidation, lipoprotein uptake and secretion are regulated by the liver, an understanding of the regulatory mechanisms that influence hepatic lipid homeostasis and systemic energy balance is of paramount importance in gaining insights that might be useful in the management of fatty liver disease. In recent years, increasing attention is being focused on certain transcription factors/nuclear receptors that are known to serve as key regulatory molecules to influence hepatic lipid synthesis, storage and oxidation. In particular, the three members of the peroxisome proliferator-activated receptor (PPAR) subfamily of nuclear receptors, namely PPARα, PPARβ/δ, and PPARγ, govern the regulation of liver lipid metabolism and thus influence the development of hepatic steatosis and fatty liver disease. Of the three members of PPAR subfamily, PPARγis critical for conserving energy as it contributes to adipogenesis, whereas both PPARαand PPARβparticipate in energy expenditure. Overexpression of PPARγin mouse liver leads to adipogenic hepatic steatosis ("hepatic adiposis") and induces the expression of adipocyte-specific and lipogenesis-related genes. In contrast, liver-specific disruption of PPARγ, exerts an opposite effect in that it dramatically reduces fatty liver. Thus, PPARγplays an important role in liver lipid metabolism and contributes to hepatic steatosis.In the nucleus, PPARs heterodimerize with retinoid X receptorα(RXRα) and bind to peroxisome proliferator response elements (PPREs) in the promoter region of target genes. Transcriptional activity of nuclear receptors and other transcription factors requires certain coactivators and coactivator-associated proteins that include MED1, SRC/p160 family of proteins, CBP/p300, PRIP, PIMT, CARM1, PRIC285, PRIC295, PRIC320, PGC-1αand others. Coactivator MED1 is a key component of Mediator complex, and is required for RNA polymerase II dependent gene transcription. Evidence indicates that MED1 is required for PPARα-mediated transcriptional activity in vivo and PPARαligand induced liver tumor. Invitro experiment displays that MED1 plays an important role in PPARγstimulated adipogenic differentiation. However, the in vivo role of MED1 and other coactivators in liver with regards to PPARγfunction remains unknown.To delineate the in vivo function of coactivator molecules in PPARγ-stimulated adipogenic hepatic steatosis, we used in this study genetically altered mouse lineages, and cellular and molecular biotechnology, including amplification and purification of adenovirus, H&E staining, Oil Red O staining, immunohistochemistry, immunofluorescence, confocal microscopy, biochemical assays of triglyceride and cholesterol, fast protein liquid chromatography (FPLC), primary hepatocyte isolation and culture, Real-time PCR, Northern Blot, Western Blot, Chromatin immunoprecipitation (ChIP) assay and microarray analysis. The reulsts demonstrate that deletion of MED1 in mouse liver impairs high fat diet and PPARγ-stimulated adipogenic steatosis, whereas deficiency of coactivators such as SRC-1, PRIC285, PRIP, and PIMT had no effect. The specific results are as follows:1. MED1 is required for PPARγ-stimulated hepatic steatosis and the expression of adipogenic genes in vivo. MED1Δliv mice injected with adenovirus-PPARγ(Ad/PPARγ) by tail vein for 6 days did not develop fatty liver, whereas MED1^fl/fl mice injected with Ad/PPARγdeveloped severe hepatic steatosis. Gene expression profiling and Northern blot analyses of Ad/PPARγinjected mouse livers showed impaired induction in MED1Δliv mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin and lipid droplet-associated genes, including caveolin-1, CideA, S3-12 and others. These adipocyte-specific and lipogenesis-related genes are strongly induced in MED1^fl/fl mouse liver in response to Ad/PPARγ. cDNA microarray analysis showed that upregulation of lipogenesis related gene networks by PPARγrequires intact MED1 gene, and in the absence of MED1 in liver the levels of expression of these genes were markedly subdued, which clearly establish that MED1 plays a key role in facilitating the transcriptional regulation of PPARγtarget genes. Furthermore, re-expression of MED1 using Ad/MED1 in MED1^ΔLiv mouse liver restored PPARγ-stimulated hepatic adipogenic response. In addition, ChIP reveals no recruitment of MED1 and slightly reduced association in MED1Δliv mouse liver of PRIP and PIMT with aP2 gene promoter, suggesting that MED1 is required for the transcriptional activation of target genes of PPARγby its ability to stabilize Mediator complex necessary for RNA polymerase II dependent transcription.2. MED1 is required for PPARγ-induced transdifferentiation of hepatocytes toward adipocytes and the expression of adipogenic genes in vitro. Primary hepatocytes isolated from MED1^ΔLiv mouse were infected with Ad/PPARγfor 12 hours. Histology and Real time PCR showed that hepatocytes from MED1^ΔLiv mouse are failed to PPARγ-stimulated hepatic adiposis and expression of adipocyte mark genes aP2 and PPARγtarget genes S3-12 and CideA.3. Other transcription coactivators SRC-1, PRIP, PIMT, and PRIC285 are dispensable for PPARγ-stimulated fatty liver development while MED1 is necessary for PPARγdependent transcription of downstream target genes and the development of hepatic steatosis. Other PPARs coactivators germ-line knockout SRC-1 (SRC-1^-/-) and PRIC285 (PRIC285^-/-) mice and liver conditional null (PRIP^ΔLiv) and (PIMT^ΔLiv) mice and their corresponding control mice were injected with Ad/PPARγand killed 5 days later. Fatty liver developed in mice lacking SRC-1, PRIC285, PRIP, and PIMT and their corresponding intact floxed controls after Ad/PPARγadministration. Northern Blot analysis revealed similar levels of increases in hepatic mRNA levels of adipogenesis genes in knockout and control mice following PPARγoverexpression.4. MED1 is required for high fat diet induced fatty liver. MED1^ΔLiv and MED1^fl/fl mice were fed high fat diet (60% kcal fat) for 0, 1, 2, 3, 4, 8 and 16 weeks. Histolocial test revealed that MED1Δliv mice when fed a high fat diet for up to 16 weeks failed to develop fatty liver, whereas MED1^fl/fl fed a high-fat diet developed severe hepatic steatosis, which was not associated with induction of PPARγtarget gene aP2. These results suggest that MED1 has significant PPARγ-independent effects on hepatic steatosis. On the other hand, PPARγ-stimulated hepatic steatosis is dependent upon MED1. Glucose and insulin tolerance tests revealed that MED1^ΔLiv mice fed a high fat diet for 4 or 16 weeks displayed lower glucose levels and exhibited greater insulin sensitivity than MED1^fl/fl mice. These results suggest that MED1 deficiency increases glucose tolerance and insulin sensitivity. In addition, MED1^ΔLiv mice showed significant elevated plasma cholesterol under short term high fat diet.5. Hepatic MED1 deficient mice showed hyperlipidemia in response to fasting. There was no fat accumulation in livers of MED1^ΔLiv mice compared to MED1^fl/fl and PPARα^-/- control mice after 72 hours of fasting. Compared with MED1^fl/fl mice, plasma triglycerides and cholesterol in MED1^ΔLiv mice were significantly increased after 24, 48 and 72 hours of fasting. FPLC showed that lipoprotein profiles were similar in fed MED1^fl/fl and MED1^ΔLiv mice. However, VLDL was significantly increased in MED1^ΔLiv mice after 24 hours of fasting, which suggests MED1 may regulate VLDL and plays a pivotal role in triglyceride and cholesterol metabolism.We conclude that transcription coactivator MED1 is required for high-fat diet-induced and PPARγ-stimulated fatty liver development in vivo, which points to a new layer of regulatory complexity in the development of hepatic steatosis and suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis.